1. Field of the Invention
The present invention relates to a method for increasing intracellular NADPH availability produced from NADH to enhance methionine production. Advantageously, the method for increasing levels of NADPH is associated to an increase of the one carbon metabolism to improve methionine production.
2. Description of Related Art
Sulphur-containing compounds such as cysteine, homocysteine, methionine or S-adenosylmethionine are critical to cellular metabolism and are produced industrially to be used as food or feed additives and pharmaceuticals. In particular methionine, an essential amino acid, which cannot be synthesized by animals, plays an important role in many body functions. Aside from its role in protein biosynthesis, methionine is involved in transmethylation and in the bioavailability of selenium and zinc. Methionine is also directly used as a treatment for disorders like allergy and rheumatic fever. Most of the methionine that is produced is added to animal feed.
With the decreased use of animal-derived proteins as a result of BSE and chicken flu, the demand for pure methionine has increased. Chemically D,L-methionine is commonly produced from acrolein, methyl mercaptan and hydrogen cyanide. Nevertheless the racemic mixture does not perform as well as pure L-methionine, as for example in chicken feed additives (Saunders on, C. L., (1985) British Journal of Nutrition 54, 621-633). Pure L-methionine can be produced from racemic methionine e.g. through the acylase treatment of N-acetyl-D,L-methionine, which increases production costs dramatically. The increasing demand for pure L-methionine, coupled to environmental concerns, render microbial production of methionine attractive.
The cofactor pairs NADPH/NADP+ and NADH/NAD+ are essential for all living organisms. They are donor and/or acceptors of reducing equivalents in many oxidation-reduction reactions in living cells. Although chemically very similar, the redox cofactors NADH and NADPH serve distinct biochemical functions and participate in more than 100 enzymatic reactions (Ouzonis, C. A., and Karp, P. D. (2000) Genome Res. 10, 568-576). Catabolic reactions are normally linked to NAD+/NADH, and anabolic reactions are normally linked to NADP+/NADPH. Together, these nucleotides have a direct impact on virtually every oxidation-reduction metabolic pathway in the cell.
Many industrially useful compounds require the cofactor NADPH for their biosynthesis, as for instance: production of ethanol on xylose in yeast (Verho et al., (2003) Applied and Environmental Microbiology 69, 5892-5897), high-yield production of (+)-catechins (Chemler et al., (2007) Applied and Environmental Microbiology 77, 797-807), lycopene biosynthesis in E. coli (Alper, H. et al., (2005) Metab. Eng. 7, 155-164) or lysine production in Corynebacterium glutamicum (Kelle T et al., (2005) L-lysine production; In: Eggeling L, Bott M (eds) Handbook of corynebacterium glutamicum. CRC, Boca Raton, pp 467-490).
The biotechnological production of L-methionine with Escherichia coli requires a sufficient pool of NADPH. Methionine is derived from the amino acid aspartate, but its synthesis requires the convergence of two additional pathways, cysteine biosynthesis and C1 metabolism (N-methyltetrahydrofolate). Asparate is converted into homoserine by a sequence of three reactions. Homoserine can subsequently enter the threonine/isoleucine or the methionine biosynthetic pathway.
Production of one molecule of L-methionine requires 8.5 moles of NADPH. To meet the NADPH requirement for both growth of E. coli and L-methionine production on minimal medium, the inventors propose here to increase the transhydrogenase activity, in the aim to increase the pool of NADPH in the cell.
The transhydrogenase reaction (below) may be catalyzed by either a membrane-bound, proton-translocating enzyme (PntAB) or a soluble, energy-independent isoform enzyme (SthA).[NADPH]+[NAD+]+[H+in]⇄[NADP+]+[NADH]+[H+out]
E. coli possesses two nicotinamide nucleotide transhydrogenases encoded by the genes sthA (also called udhA) and pntAB (Sauer U. et al., 2004, JBC, 279:6613-6619). The physiological function of the transhydrogenases PntAB and SthA in microorganisms is the generation and the re-oxidation of NADPH, respectively (Sauer U. et al., 2004, JBC, 279:6613-6619). The membrane-bound transhydrogenase PntAB uses the electrochemical proton gradient as driving force for the reduction of NADP+ to NADPH by oxidation of NADH to NAD+ (Jackson J B, 2003, FEBS Lett 545:18-24).
Several strategies have been employed to improve the availability of NADPH in whole cells. Moreira dos Santos et al. (2004) report the use of NADP+-dependent malic enzymes to increase cytosolic levels of NADPH within Saccharomyces cerevisiae. Weckbecker and Hummel (2004) describe the overexpression of pntAB in E. coli to improve the NADPH-dependent conversion of acetophenone to (R)-phenylethanol. Sanchez et al. (2006) describe the overexpression of the soluble transhydrogenase SthA in E. coli to improve the NADPH-dependent production of poly(3-hydroxybutyrate).
The inventors have surprisingly found that, by increasing PntAB activity and reducing SthA catalyzed reaction in the bacterium, the methionine production is significantly increased.
Moreover, in combining a high transhydrogenase activity and an increase of the methylenetetrahydrofolate reductase (MetF) activity, to boost the one carbon metabolism in the cell, L-methionine production by fermentation of the modified microorganisms is greatly improved.